This invention relates generally to the fields of biology, molecular biology, chemistry and biohemistry. Specifically, the invention is directed to protein-fragment complementation assays (PCAs) based on fluorescent proteins. This invention is directed to methods for the design and creation of suitable fragment pairs, to the compositions of the fragments, and to combinations suitable for PCA. Preferred embodiments include fragments of mutant fluorescent proteins having properties suitable for biotechnology applications.
The growing list of naturally fluorescent, bioluminescent or phosphorescent proteins includes GFP derived from Aequorea Victoria, and a growing number of sequence variants of GFP with useful properties. The list also includes the red fluorescent protein (RFP) derived from Discosoma; and the kindling fluorescent protein (KFP1) derived from Anemonia. These proteins are autocatalytic enzymes that are all capable of generating highly visible, efficiently emitting internal fluorophores as a result of endo-cyclization of core amino acid residues. Another common feature of the fluorescent proteins is that the signal is stable, species independent, and does not require any substrates or cofactors for the generation of a signal. These fluorescent proteins are remarkably similar structurally allowing similar principles of protein engineering to be applied across species.
The full-length DNA, and corresponding amino acid sequence of one isotype of GFP (“wild-type GFP”) is shown in TABLE 1 and has been fully described and characterized (see e.g. Tsien et al., 1998, Ann. Rev. Biochem. 67: 509–44). The intact protein (FIGS. 1 and 2B) generates a strong visible absorbance and fluorescence from a p-hydroxybenzylideneimidazolone chromophore, which is generated by cyclization and oxidation of the protein's own Ser-Tyr-Gly sequence at positions 65 to 67. Newly synthesized fluorescent protein polypeptides need to mature properly before emitting fluorescence. The maturation process involves two steps: folding and chromophore formation. First, the protein folds into a native conformation, and then the internal tripeptide cyclizes and is oxidized. In this regard the fluorescent protein is an enzyme which autocatalyzes the cyclization reaction, requiring only molecular oxygen for completion of the reaction.
A variety of useful mutant versions of the full-length, wild-type GFP have been generated and have been termed ‘Aequorea fluorescent protein (AFP) variants’ or AFPs. These “mutant fluorescent proteins” have proven to have wide applicability for biology and biotechnology applications as a result of their improved spectral properties. Some of the reported GFP variants are shown in Table 2. By conventional usage, the positions of the mutations (as in Table 2 and throughout this invention) are denoted relative to the sequence of wild-type GFP (Table 1). Many of these AFPs exhibit vastly improved properties over the original wild-type GFP in terms of signal intensity, generating a fluorescence signal 5 to 30 times that of the wild-type protein. The enhanced GFP (EGFP), which is the basis for nearly all biology applications and for mutant fluorescent proteins, has improved codon usage for mammalian cells.
Starting with GFP, mutations at the site of the chromophore have been created which result in different color variants. Mutations of the side chains in contact with the chromophore have been shown to further enhance protein folding and brightness. Combinations of mutations have been created that have spectral shifts and that fold more rapidly at 37° C., producing brighter signals for cell biology applications. The most common spectral variants include the widely-used yellow (YFP/EYFP), cyan (CFP/ECFP) and BFP variants (R. Y. Tsien, 1998, “The Green Fluorescent Protein”, in: Annual Reviews of Biochemistry 67: 509–544).
Additional mutants of GFP have been created with unique properties. These include a ‘CGFP’ variant with an excitation and emission wavelength intermediate between CFP and EGFP (J. Zhang et al., 2000, “Creating new fluorescent probes for cell biology”, Nature Reviews 3: 906–918). The ‘citrine’ variant of YFP (YFP-Q69M) confers a lower pKa than for previous YFPs, indifference to chloride anion, twice the photostability of previous YFPs, and much better expression at 37° C. and in organelles (0. Griesbeck et al., 2001, “Reducing the Environmental Sensitivity of Yellow Fluorescent Protein”, J. Biol. Chem 276: 29188–29194).
Several versions of YFP have been created using random mutagenesis. These mutant proteins have fluorescence intensities 3–30 times brighter than EYFP. They include the so-called super-EYFP (SEYFP) (EYFP—F64L/M153T/V163A/S175G) and ‘Venus’ (SEYFP-F46L) (T. Nagai et al., 2002, “A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications”, Nature Biotech. 20: 87–90). Venus contains the novel mutation, F46L, which at 37° C. greatly accelerates oxidation of the chromophore, the rate-limiting step of mutation. As a result of the additional SEYFP mutations, Venus SEYFP-F46L also folds well and is relatively tolerant of exposure to acidic or high chloride anion concentrations.
A photoactivatable form of GFP named PA-GFP (GFP-V163A/T203H) has been reported that, after intense irradiation with 413-nm light, increases fluorescence 100 times when excited by 488-nm light and remains stable for days under aerobic conditions (G. H. Patterson & J. L.-Schwartz, “A photoactivatable GFP for selective photolabeling of proteins and cells”, Science 297: 1873–1877, 2002).
TABLE 1Full-length Aequorea GFP nucleic acid sequence (716 bp) (SEQ ID No:1) andcorresponding amino acid sequence (238 aa) (SEQ ID No:2). Amino acids arenumbered at every 5th position. This sequence is for the wild-type protein. In“enhanced” versions of GFP (EGFP, EYFP, ECFP) a valine residue is insertedafter the initiating methionine. The valine becomes amino acid # 2 and theremaining amino acids are shifted accordingly. Descriptions of GFP mutants(as in Table 2 and throughout the specifications) refer to the numbering shownbelow. Alternative fragmentation sites that are the subject of the presentinvention are shown at the following regions (underlined) amino acid residues38–40 (region 1); residues 101–103 (region 2); residues 114–118 (region 3);residues 154–160 (region 4); residues 171–175 (region 5); and residues 188–190(region 6). The positions of specific amino acid residues are shown forTyrosine 39 (Y39), Aspartate 102 (D102), Glutamine 157 (Q157), Lysine 158(K158), Aspartate 173 (D173) and Aspartate 190 (D190).atg  agt  aaa  gga  gaa  gaa  ctt  ttc  act  gga  gtt  gtc  cca  att  ctt  gttMet  Ser  Lys  Gly  Glu  Glu  Leu  Phe  Thr  Gly  Val  Val  Pro  Ile  Leu  Val1                   5                        10                       15 gaa  tta  gat  ggt  gat  gtt  aat  ggg  cac  aaa  ttt  tct  gtc  agt  gga  gagGlu  Leu  Asp  Gly  Asp  Val  Asn  Gly  His  Lys  Phe  Ser  Val  Ser  Gly  Glu               20                       25                       30 ggt  gaa  ggt  gat  gca  aca  tac  gga  aaa  ctt  acc  ctt  aaa  ttt  att  tgcGly  Glu  Gly  Asp  Ala  Thr  Tyr  Gly  Lys  Leu  Thr  Leu  Lys  Phe  Ile  Cys          35                  Y39  40                       45 act  act  gga  aaa  cta  cct  gtt  cca  tgg  cca  aca  ctt  gtc  act  act  ttcThr  Thr  Gly  Lys  Leu  Pro  Val  Pro  Trp  Pro  Thr  Leu  Val  Thr  Thr  Phe     50                       55                       60 tct  tat  ggt  gtt  caa  tgc  ttt  tca  agc  tac  cca  gat  cat  atg  aaa  cggSer  Tyr  Gly  Val  Gln  Cys  Phe  Ser  Arg  Tyr  Pro  Asp  His  Met  Lys  Arg65                       70                       75                       80 cat  gac  ttt  ttc  aag  agt  gcc  atg  ccc  gaa  ggt  tat  gta  cag  gaa  agaHis  Asp  Phe  Phe  Lys  Ser  Ala  Met  Pro  Glu  Gly  Tyr  Val  Gln  Glu  Arg                    85                       90                       95 act  ata  ttt  ttc  aaa  gat  gac  ggg  aac  tac  aag  aca  cgt  gct  gaa  gtcThr  Ile  Phe  Phe  Lys  Asp  Asp  Gly  Asn  Tyr  Lys  Thr  Arg  Ala  Glu  Val               100       D102           105                      110 aag  ttt  gaa  ggc  gat  acc  ctt  gtt  aat  aga  atc  gag  tta  aaa  ggt  attLys  Phe  Glu  Gly  Asp  Thr  Leu  Val  Asn  Arg  Ile  Glu  Leu  Lys  Gly  Ile          115  G116                120                      125 gat  ttt  aaa  gaa  gat  gga  aac  att  ctt  gga  cac  aaa  ttg  gaa  tac  aacAsp  Phe  Lys  Glu  Asp  Gly  Asn  Ile  Leu  Gly  His  Lys  Leu  Glu  Tyr  Asn     130                      135                      140 tat  aac  tca  cac  aat  gta  tac  atc  atg  gca  gac  aaa  caa  aag  aat  ggaTyr  Asn  Ser  His  Asn  Val  Tyr  Ile  Met  Ala  Asp  Lys  Gln  Lys  Asn  Gly145                      150                      155       Q157 K158      160 atc  aaa  gtt  aac  ttc  aaa  att  aga  cac  aac  att  gaa  gat  gga  agc  gttIle  Lys  Val  Asn  Phe  Lys  Ile  Arg  His  Asn  Ile  Glu  Asp  Gly  Ser  Val                    165                      170            D173      175 caa  cta  gca  gac  cat  tat  caa  caa  aat  act  cca  att  ggc  gat  ggc  cctGln  Leu  Ala  Asp  His  Tyr  Gln  Gln  Asn  Thr  Pro  Ile  Gly  Asp  Gly  Pro               180                      185                      D190 gtc  ctt  tta  cca  gac  aac  cat  tac  ctg  tcc  aca  caa  tct  gcc  ctt  tcgVal  Leu  Leu  Pro  Asp  Asn  His  Tyr  Leu  Ser  Thr  Gln  Ser  Ala  Leu  Ser          195                      200                      205 aaa  gat  ccc  aac  gaa  aag  aga  gac  cac  atg  gtc  ctt  ctt  gag  ttt  gtaLys  Asp  Pro  Asn  Glu  Lys  Arg  Asp  His  Met  Val  Leu  Leu  Glu  Phe  Val     210                      215                      220 aca  gct  gct  ggg  att  aca  cat  ggc  atg  gat  gaa  cta  tac  aaaThr  Ala  Ala  Gly  Ile  Thr  His  Gly  Met  Asp  Glu  Leu  Tyr  Lys225                      230                      235
TABLE 2Spectral characteristics of the major classes ofAequorea fluorecent proteins (AFPs)CommonRel. fl.Mutationname□exc(ε)□cm(QY)@ 37° C.Class 1, wild-typeNone or Q80RWild type395–397(25–30)504(0.79)6470–475(9.5–14)F99S, M153T, V163ACycle 3397(30)506(0.79)100475(6.5–8.5)Class 2, phenolate anionS65T489(52–58)509–511(0.64)12F64L, S65TEGFP488(55–57)507–509(0.60)20F64L, S65T, V163A488(42)511(0.58)54S65T, S72A, N149K,Emerald487(57.5)509(0.68)100M153T, I167TClass 3, neutral phenolS202F, T203IH9399(20)511(0.60)13T203I, S72A, Y145FH9-40399(29)511(0.64)100Class 4, phenolate anionwith stacked π-electronsystem (yellow fluorescentproteins) (YFPs)S65G, S72A, T203F512(65.5)522(0.70)6S65G, S72A, T203YEYFP508(48.5)518(0.78)12S65G, V68L, Q69K10C Q69K516(62)529(0.71)50S72A, T203YS65G, V58L, S72A, T203Y10C514(83.4)527(0.61)58S65G, S72A, K79R,Topaz514(94.5)527(0.60)100T203YF46LEYFP-F46L515(78.7)528(0.61)NDF64L, M153T, V163A, S175GSEYFP515(101)528(0.56)NDF46L, F64L, M153T, V163A,SEYFP-F46Lsee: Nagai et al., NatureS175G(‘Venus’)Biotech. 20: 87–90, 2002V68L, Q69M‘Citrine’see: Griesbeck et al.,J. Biol. Chem 276:29188–29194 (2001)V163A, T203HPA-GFPsee: Patterson et al.,Science 297: 1873–1877, 2002Class 5, indole inchromophore (cyanfluorescent proteins)(CFPs)Y66W436485—Y66W, N146I, M153T,W7434(23.9)476(0.42)61V163A452505F64L, S65T, Y66W,W1B or434(32.5)476(0.4)80N146I, M153T, V163AECFP452505S65A, Y66W, S72A,W1C435(21.2)495(0.39)100N146I, M153T, V163AT203YCGFPsee: Sawano & Miyawaki, NucleicAcid Res. 28: E78 (2000)Class 6, imidazolein chromophore(blue fluorescentproteins) (BFPs)Y66HBFP384(21)448(0.24)18Y66H, Y145FP4-3382(22.3)446(0.3)52F64L, Y66H, Y145FEBFP380–383(26.3–31)440–447(0.17–0.26)100Class 7, phenyl inchromophoreY66F360442—
TABLE 3Alignment of wild type Aequorea victoria GFP and Aequorea-derivedfluorescent proteins (Zhang et al. 2002). New variants of greenfluorescent protein (GFP) (SEQ ID No:2) that encode proteins withaltered excitation and emission wavelength properties relative to wildtype GFP are aligned. These include the mammalian codon-usage optimizedECFP (cyan) (SEQ ID No:8), EGFP (green) (SEQ ID. No:3), (cyan), EGFP(green), and EYFP (yellow) (SEQ ID No:4) variants. Three more recentvariants of EYFP include EYFP-Q69M (Citrine) (SEQ ID No:5), super-EYFP(SEYFP) (SEQ ID No:6), and SEYFP-F46L (‘Venus’) (SEQ ID No:7).GFP  1-MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTEGFP  1MV..........................................................EYFP  1MV..........................................................EYFP-Q69M  1MV..........................................................SEYFP  1MV..........................................................SEYFP-F46L  1MV............................................L.............ECFP  1MV.......................................................... GFP 60LVTTFSYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLEGFP 61....LT......................................................EYFP 61.....G..L...A...............................................EYFP-Q69M 61.....G..LM..A...............................................SEYFP 61....LG..L...A...............................................SEYFP-F46L 61....LG..L...A...............................................ECFP 61....LTW..................................................... GFP120VNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLAEGFP121............................................................EYFP121............................................................EYFP-Q69M121............................................................SEYFP121.................................T.........A...........G....SEYFP-F46L121.................................T.........A...........G....ECFP121..........................I......T.........A................ GFP180DHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYKEGFP181...................................................L.......EYFP181.......................Y...........................L.......EYFP-Q69M181.......................Y...........................L.......SEYFP181.......................Y...........................L.......SEYFP-F46L181.......................Y...........................L.......ECFP181...................................................L.......Fluorescent proteins from species other than Aequorea victoria have also been isolated and characterized. The growing list includes a green fluorescent protein from Renilla reniformis, and a number of fluorescent proteins from the coral Anthozoa. These include the red fluorescent protein from Discosoma (DsRed) (M. V. Matz et al., 1999, Nature Biotech. 17:969–973) which has been crystallized (Yarbrough et al., 2001, Proc. Natl. Acad. Sci. 98: 462–467) and has found wide applicability as a biology tool. Although the coral fluorescent proteins have only 26–30% sequence identity with Aequorea GFP, they are remarkably similar structurally. In particular, the coral fluorescent proteins share the same β-can fold first observed in GFP. All the key secondary structure elements observed in GFP could be easily detected in the coral proteins in the same arrangements, and remarkable similarity was observed in the stretches forming the “caps' of the can. Key residues thought to be involved in chromophore formation in GFP are also conserved in the coral proteins, including an Arginine at residue 96, the Tyrosine at residue 66 and Glycine at residue 67.
The structural homology of fluorescent proteins among various species means that many of the principles of genetic engineering and protein engineering previously applied to GFP can also be applied to these fluorescent proteins to create variants with desirable properties for biological applications and biotechnology.
The availability of a bright orange-red fluorescent protein with a high quantum yield would be particularly useful for biological studies as it is spectrally distinct from the previously described green, yellow and cyan variants of GFP. DrFP583, commonly known as DsRed, is a 28-kDa polypeptide that has essentially the same chromophore as GFP, which is autocatalytically formed from an internal Gln-Tyr-Gly (residues 66–68) tripeptide. DsRed is remarkably similar structurally to A. victoria GFP. In fact, the overall fold of DsRed is virtually identical to GFP, consisting of a slightly irregular 11-stranded beta-barrel (described as a beta can) with a coaxial central helix and alpha-helical caps on the barrel ends. The sequence alignment of the coral fluorescent proteins with Aequorea GFP is shown in Table 4.
A number of mutant versions of DsRed have now been described with faster rates of chromophore maturation than the wild-type protein (B. J. Bevis and B. S. Glick, Nature Biotech. 20: 83–86, 2002). Importantly, DsRed has recently been engineered into a monomeric form (mRFP) (R. E. Campbell et al., Jun. 11, 2002, “A monomeric red fluorescent protein”, Proc. Natl. Acad. Sci. 99(12): 7877–7882) which is more useful than the multimeric protein as a reporter. mRFP1 is a monomer, the signal matures >10-fold faster than for DsRed, and the monomeric protein has minimal emission at wavelengths suitable for excitation of GFP.
A unique GFP-like chromoprotein asCP from the sea anemone Anemonia sulcata was recently discovered (Chudakov, D. M., et al. 2003, Kindling fluorescent proteins for precise in vivo photolabeling”. Nat. Biotechnol. 21, 191–194). asCP is initially nonfluorescent, but in response to intense green light irradiation it becomes brightly fluorescent (kindles) with emission at 595 nm. Kindled asCP relaxes back to the initial nonfluorescent state with a half-life of <10 seconds. Alternatively, fluorescence can be “quenched” instantly and completely by a brief irradiation with blue light. A mutant (asCP A148G, or KFP1) has been generated which is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form that has 30 times greater fluorescent intensity than the unkindled protein. This “kindling fluorescent protein” can be used for precise in vivo photolabeling to track the movement of cells, organelles and proteins.
Fluorescent proteins have proven to be useful reporters for monitoring gene expression and protein localization in vivo and in real time (J. M. Tavare et al., 2001, J. Endocrinol. 170: 297–306; Thastrup et al., U.S. Pat. No. 6,518,021). Such assays measure cellular events linked to individual proteins, as compared with binary or higher-order events. A number of other useful applications of fluorescent proteins have been described, including the construction of biochemical sensors and the creation of innovative fusion constructs to analyze protein dynamics in living cells. For the measurement of bimolecular events, FRET (fluorescence resonance energy transfer) or BRET (bioluminescence resonance energy transfer) assays have been well described (A. Miyawaki & R. Tsien, 2000, Methods in Enzymology 327: 472–500; G. W. Gordon et al., 1998, Biophys. J. 74: 2702–2713). GFP, BFP, CFP and RFP have been used in FRET or BRET assays to detect protein-protein interactions, monitor protease activity, and create calcium indicators, among other uses.
It is important to note that all the above-mentioned applications rely upon tagging of proteins of interest with a functional, full-length (or substantially full-length) fluorescent protein (lumiphore). None of the references cited above describe compositions or uses of fragments of fluorescent proteins.
Protein-fragment complementation assays (PCA) represent a general method for the construction of assays for the detection and quantitation of biomolecular and drug interactions (J. N. Pelletier, J. N., Remy, I. and Michnick, S. W. 1998, Protein-Fragment complementation Assays: a general strategy for the in vivo detection of Protein-Protein Interactions, J. Biomolecular Techniques 10:32–19; Remy, I., Pelletier, J. N., Galarneau, A. & Michnick, S. W. 2002, Protein Interactions and Library Screening with Protein Fragment Complementation Strategies, in: Protein-protein Interactions: A Molecular Cloning Manual, Cold Spring Harbor Laboratory Press Chapter 25, 449–475; Michnick, S. W., Remy, I., C.-Valois, F. X., Vallee-Belisle, A., Galarneau, A. & Pelletier, J. N., 2000, Detection of Protein-Protein Interactions by Protein Fragment Complementation Strategies, Parts A and B, in: Methods in Enzymology 328:208–230.; J. N. Pelletier & S. W. Michnick., 1997, A Strategy for Detecting Protein-Protein Interactions in vivo Based on Protein Fragment Complementation. Protein Engineering, 10(Suppl.): 89.).
PCA involvse the oligomerization-assisted complementation of fragments of a reporter protein such as a monomeric enzyme, a fluorescent protein, luminescent protein or phosphorescent protein. Dimeric and multimeric enzymes can also be used in PCA, however, monomeric proteins are preferred. As described by Michnick et al. (U.S. Pat. No. 6,270,964) the ideal properties of a protein suitable for PCA are: 1) a protein or enzyme that is relatively small and monomeric; 2) for which there is a large literature of structural and functional information; 3) for which simple assays exist for the reconstitution of the protein or activity of the enzyme; and 4) for which overexpression in eukaryotic and prokaryotic cells has been demonstrated.
FIG. 1 of U.S. Pat. No. 6,270,964 shows a general description of a PCA. The gene for a protein or enzyme is rationally dissected into two or more fragments. Using molecular biology techniques, the chosen fragments are subcloned, and to the 5′ ends of each, proteins that either are known or thought to interact are fused. Co-transfection or transformation of these DNA constructs into cells is then carried out. Reassembly of the probe protein or enzyme from its fragments is catalyzed by the binding of the test proteins to each other, and reconstitution is observed with some assay. It is crucial to understand that these assays will only work if the fused, interacting proteins catalyze the reassembly of the protein or enzyme. That is, observation of reconstituted protein or enzyme activity must be a measure of the interaction of the fused proteins.
U.S. Pat. No. 6,270,964 taught the principles, methods and applications of PCAs for a large number of useful reporters that can generate a fluorescent signal (see Table 1). Example 3 of that patent describes various embodiments of PCAs including a number of specific reporters suitable for PCA. Details were described for glutathione-S-transferase, firefly luciferase, xanthine-guanine phosphoribosyl transferase (XPRT), diaphorase, adenosine deaminase, bleomycin binding protein, hygromycin-B-phosphotransferase, histidinol NAD+oxidoreductase and Aequorea green fluorescent protein (GFP). Table 1 of U.S. Pat. No. 6,270,964 described an even larger list of other reporters meeting the criteria for PCA.
In Example 3 of U.S. Pat. No. 6,270,964 a PCA based on GFP was described including its properties and advantages: “GFP from Aequorea victoria is becoming one of the most popular protein markers for gene expression. This is because the small, monomeric 238 amino acids protein is intrinsically fluorescent due to the presence of an internal chromophore that results from the autocatalytic cyclization of the polypeptide backbone between residues Ser65 and Gly67 and oxidation of the bond of Tyr 66. The GFP chromophore absorbs light optimally at 395 nm and possesses also a second absorption maximum at 470 nm. This bi-specific absorption suggests the existence of two low energy conformers of the chromophore whose relative population depends on the local environment of the chromophore. A mutant Ser65Thr that eliminates isomerization results in a 4 to 6 times more intense fluorescence than the wild type. Recently the structure of GFP has been solved by two groups, making it a candidate for a structure-based PCA design which we have begun to develop. As with the GST assay we are doing all of our initial development in E. Coli with GCN4 leucine zipper-forming sequences as oligomerization domains. Direct detection of fluorescence by visual observation under broad spectrum UV light will be used. We will also test this system in COS cells, selecting for co-transfectants using fluorescence activated cell sorting.” The issued claims of U.S. Pat. No. 6,270,964, U.S. Pat. No. 6,294,330 and U.S. Pat. No. 6,428,951 include fluorescent proteins in addition to other reporter classes. PCAs have been used to screen diverse peptide libraries (J. N. Pelletier, et al., 2000, Nature Biotech. 17: 683–690) and cDNA or antibody libraries (E. Moessner et al., 2001, J. Mol. Biol. 308: 115–122; I. Remy et al., submitted for publication); to quantify the association constants of protein domains such as parallel and anti-parallel leucine zipper-forming sequences (K. M. Arndt et al., 2000, J. Mol. Biol. 295: 627–639; I. Ghosh et al., 2000, J. Am. Chem. Soc 122:5658–5659); to detect the drug-induced association and dissociation of protein complexes (I. Remy and S. W. Michnick, 1999, Proc Natl Acad Sci USA 96: 5394–5399); to measure the ligand-induced activation of cellular receptors (I. Remy et al., 1999, Science 283: 990–993); to study transcription factor complexes in live cells (R. Subramaniam et al., 2001, Nature Biotech. 19: 769–772, 2001); to quantitate elements of signal transduction pathways in real time (I. Remy and S. W. Michnick, 2001, Proc Natl Acad Sci USA, 98: 7678–7683, 2001; A. Galarneau et al., 2002, Nature Biotech. 20: 619–622); and to pinpoint the subcellular locations of protein-protein complexes (I. Remy and S. W. Michnick, 2001, Proc Natl Acad Sci USA 98: 7678–7683; R. Subramaniam et al., 2001, Nature Biotech., 19: 769–772; C.-D. Hu et al., Molecular Cell 9: 789–798, 2002; H. Yu et al., submitted for publication).
Subsequent to our inventions describing the use of GFP for PCA, Ghosh et al. (J. Am. Chem. Soc 122:5658–5659, 2000; US 2002/0146701) used a GFP PCA to study GCN4 leucine zipper oligomerization in a manner originally proposed by Michnick et al. They showed antiparallel leucine zipper-directed reassembly of GFP fragments in bacteria. A single GFP variant was chosen for these studies and a single fragmentation site was used. The authors did not disclose additional principles or methods for fragmenting a fluorescent protein based on rational design beyond the principles first described in Michnick et al. (e.g. U.S. Pat. No. 6,270,964). Moreover, other than the fragment pair used in the leucine zipper study, Ghosh and coworkers did not disclose specific assay compositions useful for PCA.
Hu et al. (Molecular Cell 9: 789–798, 2002) described a PCA based on a yellow variant of GFP, where the fragments of YFP were fused either to parallel leucine zippers or to Rel family proteins. However, additional principles and methods of engineering fluorescent proteins, and fragment compositions, were not described by Hu and coworkers. Moreover, the prior art is silent on the topic of whether mutations known to affect the properties of intact fluorescent proteins would confer similar properties on polypeptide fragments used for PCA.
Since fluorescent protein PCAs do not depend upon external cofactors or substrates for signal generation, they are particularly useful for the construction of cell-based assays. A suite of fluorescent protein PCAs would enable a large number of useful assays with differing spectral properties. For example, fluorescent proteins with high quantum yields could be engineered into PCA fragments to allow detection of rare events within cells, such as complexes between proteins expressed at very low levels, or low-affinity complexes between enzymes and their substrates. In addition, PCAs with red-shifted emissions would provide improved signal to noise relative to cellular autofluorescence which often occurs in the green channel. Importantly, fragments generating different color PCAs could be combined to allow simultaneous monitoring of two, three, or more cellular events (multicolor PCA). Finally, fluorescent protein PCAs could be used to create multicolor arrays for rapid diagnostics. For example, multicolor arrays based on antibodies binding to different antigens would allow the rapid and simultaneous detection of bio-warfare agents.